Co-immunoprecipitation of flotillin-2 by a PSGL-1 antibody.

<p>Cells were incubated either in Gey's medium for 30 min at 37°C (ctrl) or were stimulated with 1 nM fNLPNTL (HP) for 10 min after a 20 min preincubation. Cells were then lysed and subjected to immunoprecipitation (IP), using a murine PSGL-1 antibody or a murine IgG as control, as indicated. Upper parts of the immunoblots (IB) were probed for PSGL-1 (A) (murine Ab) and lower parts of the blots for flotillin-2 (B) (flot2, murine Ab; left upper and lower panels in B). The band of approximately 50 kD detectable in the immunoprecipitates just above flotillin (upper panels in B) corresponds to the heavy chain of IgG, as it also appeared when the blot was stripped and decorated only with the secondary anti-murine IgG antibody (upper right panel in B). Three percent input lysates were in addition analyzed for the presence of PSGL-1 (A) and flotillin-2 (B) showing that equal amounts of these proteins were present in the lysates of resting and stimulated cells before immunoprecipitation (lower panels in A and B). Data representative of 3 independent experiments are shown.